The market share of biopharmaceuticals is rapidly increasing. Over 1000 new biopharmaceutical products are in the development pipeline globally. The growth of the biopharmaceuticals market is thus driving a need for increased manufacturing capacity. However, the construction of new biomanufacturing facilities is very expensive, ranging from $50 million for a small pilot plant to over $500 million or more for a commercial facility. In addition, it takes over 4 years to bring new facilities on line, as regulatory authorities must validate all aspects of the manufacturing process.
The initial selection of host cell also influences the design of the downstream process and the number of required unit operations, such as number of bioreactor runs. Thus, the efficiency of production of any one product will translate into reduced manufacturing volume or capacity requirement and therefore overall reduced costs. One basic method by which this can be achieved, is by the creation of host cell lines that produce stable molecules and at a high rate of expression.
A number of methods have been used to increase the expression of therapeutic proteins including therapeutic monoclonal antibodies (Abs) in various mammalian cell hosts (reviewed in Wurm, et al. 2004 Nat. Biotechnol. 22, 1393-1398; Ganguly and Jin, 2002 and Trill, 2002). These include: a) the use of strong viral promoters, e.g., human cytomegalovirus promoter (Deans et al., 1984) or cellular promoter e.g., the beta-actin promoter (Niwa et al., 1991); b) the amplification of transfected genes using dihydrofolate reductase (DHFR) gene as an amplifiable marker (Kaufman, 1990) both in DHFR-negative Chinese hamster ovary cell lines (Page and Sydenham, 1991) and in myeloma cell lines (Dorai and Moore, 1987); c) the use of the glutamine synthase selection system (Bebbington et al., 1992); and d) the use of dicistronic vectors where the amplifiable marker is expressed at much lower levels compared to the recombinant protein of interest, and therefore amenable to a high degree of gene amplification and expression (Lucas et al., 1996; Kaufman et al., 1991). In addition, the use of targeted integration of the gene of interest to chromosomal ‘hot spots’ of expression (Aldrich et al., 2003 and Koduri et al., 2001) and the use of insulator sequences and anti-repressor elements in the expression vector (Zahn-Zabal et al., 2001; Kwacks et al., 2003) have yielded promising results, although their use in therapeutic protein expression is yet to be realized. Due to these and many other improvement efforts, cell lines that express therapeutic proteins with specific productivity as high as 100 picograms/cell/day have been developed (Page and Sydenham, 1991).
In order to increase the worldwide capacity for manufacturing protein products using existing facilities, cell lines with even higher productivity than those that are currently available need to be developed. Methods for screening to select such a cell line would be of utility in expediting the process of developing high expressing cell lines for manufacture of therapeutic proteins.